Haplotypic analysis of cox1 from Toxocara canis demonstrates five distinct clades that are not geographically defined

Background Toxocara canis is a cosmopolitan parasite of dogs that is transmitted transplacentally to puppies resulting in widespread shedding of eggs in the environment. However, it is not clear if there are dominant parasite genotypes that are more common, pathogenic, or likely to be zoonotic. Methods/principle findings Sequences of mitochondrial cox1 gene from adult worms were used to compare parasites from the United States with submitted sequences from parasites isolated from dogs in different countries. Our analysis revealed at least 55 haplotypes. While we expected the North American worms to form a distinct cluster, we found haplotypes of T. canis reported elsewhere existing in this population. Interestingly, combining the sequence data from our study with the available GenBank data, analysis of cox1 sequences results in five distinct clades that are not geographically defined. Conclusions The five clades of T. canis revealed in this study potentially have unique life histories, traits, or host preferences. Additional investigation is needed to see if these distinct clades represent cryptic species with clinically useful attributes or genotypes with taxonomic value. Evaluation of common mitochondrial genes may reveal distinct populations of zoonotic T. canis.


Introduction
Toxocara canis is a common nematode parasite of canines throughout the world.Adult worms live in the small intestine of dogs and lay eggs that are passed in feces.Upon ingestion of the larvated egg by susceptible dogs, larvae hatch and undergo a blood-liver-lung migration prior to being adult nematodes.However, a proportion of larvae migrate into the somatic tissues where they become arrested as hypobiotic larvae.These tissue-dwelling larvae are reactivated during the third trimester of pregnancy, crossing the placenta to infect puppies.This mechanism of ubiquitous infection has led to a widespread need for anthelmintic treatment in dogs.Toxocariasis is designated as one of five Neglected Parasitic Infections by the CDC based the number of infections, severity of illness, and the ability to prevent and/or treat infections [1].In humans, migrating larvae cause visceral and ocular larval migrans [2].A 2018 study estimated that 5% of the U.S. population is seropositive for Toxocara antibodies [3].
While T. canis is a common parasite of domestic dogs worldwide, there is relatively little data available on the population genetic structure of this organism.Understanding the phylogenetic relationship of pathogens can be important for clinical purposes as it may provide information about drug resistance, virulence, or other relevant parameters [4,5].In similar nematodes such as Ascaris suum, molecular epidemiological studies have utilized nuclear and mitochondrial sequences to investigate population structuring and haplotypic variation [6][7][8][9].For example, studies of A. suum have revealed geographic clustering of sequence relationships and potential zoonotic transmission events [6].However, few or no studies have been undertaken to describe such population traits in T. canis in the United States.
A recent study used the cytochrome c oxidase subunit 1 mitochondrial gene (cox1) to examine the relationship between T. canis, T. cati, T. malaysiensis, and Toxascaris leonina from 8 countries: Brazil, China, Denmark.Germany, Japan, Malaysia, Portugal, and Russia [10].Fava et al found that sequences grouped based on host species (canids and felids) but did not exhibit any geographic relationship.The authors suggest gene flow among populations due to global movement of animals as the reason for the lack of geographic clustering of specimens [10].
In the present study, we aimed to understand genetic and haplotypic diversity of T. canis adults isolated from dogs in the United States using the partial mitochondrial barcoding cox1 PLOS NEGLECTED TROPICAL DISEASES gene and compare it with those reported globally.We hypothesized that the geographical isolation of North America would drive haplotypic differentiation and structuring in T. canis populations in the U.S. and those reported from other global regions.

Ethics statement
The research was approved by the Iowa State University Institutional biosafety committee #21289.

Parasites
Toxocara canis adult specimens were collected by licensed veterinarians from dogs in Alabama (28 samples), Florida (14 samples), Iowa (8 samples), and Louisiana (19 samples) from 2017-2019.Species identity of the parasites were established using morphological features as identified by practicing veterinary parasitologists (the authors) and submitted to the Iowa State University College of Veterinary Medicine.The samples were collected opportunistically; any morphologically bona fide Toxocara from a dog was eligible for inclusion in the study.When possible, efforts were made to collect only 1 worm per canine host in order to search for greater genetic diversity.Parasites were identified using traditional keys [11] and stored in 70% ethanol until the time of DNA extraction.All protocols were approved by the Institutional Biosafety Committee at Iowa State University 15-I-0027-A/H.
Sterile surgical blades were used to remove a 5 mm portion of tissue from the anterior end of each worm.DNA was extracted using the Qiagen DNeasy Blood and Tissue kit (Valencia, CA) according to manufacturer's instructions.Genomic DNA was eluted in 100 μL of water and stored at -20˚C.

Phylogenetic and haplotypic analysis
A total of 63 new sequences from this study were acceptable for phylogenetic and haplotypic analysis.Additionally, sequences from Fava et al (2020) [10] and others available on GenBank were included.Blast searching, searching of FASTA files from Toxocara transcriptomes, and multiple sequence alignment was used to identify potential sequences.All sequences available on GenBank consisting of the 327 paired nucleotides were included in the analysis.The final dataset consisted of 135 sequences with 63 isolates from the United States, 1 from Australia, 12 from Brazil, 6 from China, 11 from Denmark, 5 from Germany, 24 from Iran, 1 each from Japan, Netherlands, Poland, Portugal and 9 from Russia.Sequences were trimmed to 327 base pairs and alignment was performed using ClustalW on MegaX [13].
Mega-X [13] was used to infer evolutionary history and generate maximum likelihood trees using the General Time Reversible model with a gamma distribution [14] and 1000 bootstrap replications.Toxocara cati (Accession number: MT942618.1)was used as an outgroup.Trees were constructed both with and without sequences from previous studies [10] and other T. canis cox1 sequences available on GenBank.

Identification of T. canis and sequence analysis
All PCR reactions generated an amplicon of the expected size except 4 (1 Florida, 3 Iowa) which were excluded from the analysis.NCBI BLAST analysis was used to confirm sequences were consistent with T. canis.One specimen (Iowa) blasted as Baylisascaris and was removed from the remainder of the analysis.Additionally, one specimen (Alabama) was removed from the analysis due to poor sequence quality.

Phylogenetic analysis of cox1 sequences
A maximum likelihood tree constructed using the sequences collected in this study as well as a tree constructed using additional sequences available in GenBank, including those from Fava et al [10] (GenBank accession numbers: MT359256 -MT359318), are shown in Figs 1 and 2, respectively.When analyzed on their own, the sequences from our study appeared to form loose clusters primarily made up of samples from Alabama, Florida, and Louisiana while samples from Iowa shared common characteristics from several other states (Fig 1).On the other hand, unexpectedly, when a global analysis was performed on all sequences in GenBank, samples from different continents clustered together (Fig 2 ).A maximum likelihood analysis using amino acid sequences yielded similar results (S1 and S2 Figs).

Haplotype analysis of cox1 sequences
Haplotype analysis of 135 sequences revealed 55 haplotypes with a haplotypic diversity of 0.932, nucleotide diversity of 0.01227, and 54 segregating variant sites (Figs 3 and 4).We designated the predominant haplotype as haplotype 1. Table 1 provides descriptions of the haplotypes including haplotype frequency, sequences included in each haplotype, and the country of origin for each sequence.Haplotypes demonstrated geographic grouping, with the majority (50 out of 55) of haplotypes consisting of sequences from one location.Haplotype 1 contains sequences from the U.S. (Alabama, Florida, Louisiana), Denmark, Russia, Iran, Poland and Netherlands.Haplotypes 3 and 5 were the only other shared haplotype between the U.S. and Europe.Haplotypes 4, 6-25 were novel and unique to the U.S. Haplotype 4 consisted entirely of sequences from Alabama; haplotype 9 of sequences entirely from Florida and haplotype 19 of sequences from Louisiana (Table 1 and Fig 4).A maximum likelihood analysis using amino acid sequences yielded similar results (S3).
Genetic differentiation between U.S. sequences and non-U.S.global sequences was low with an Fst of 0.08309.Fst between U.S. sequences and European, Asian, and South American sequences were 0.0569, 0.12218, and 0.12728 respectively.Distance between US sequences and Australian sequences were could not be calculated due to the paucity of data (1 sequence) from Australia.
A haplotype consensus tree was constructed using MegaX and is shown in Fig 3 .This analysis also supported the conclusion that haplotypic clustering was not geographically driven and  Interestingly, when evaluating the median joining network, we also observed 5 main clusters (Fig 4).We found that the majority of haplotypes (51/55) group in the same clusters in the median joining network as when analyzed in the maximum likelihood tree, but these relationships were not geographically defined.

Discussion
Toxocara canis is an important parasite due to its impact on both animal and human health.In this study, we investigated the haplotypes of T. canis in the United States and compared them to the haplotypic sequences available for this parasite worldwide.The evolutionary/genetic relationship of parasites can serve as a framework for understanding trends in anthelmintic resistance, virulence, host predilection, and zoonotic potential.For example, phylogenetic and haplotypic analysis was used to infer the zoonotic potential of the zoonotic parasite Strongyloides stercoralis.Genes, including Cox1, revealed two lineages of S. stercoralis, whereby lineage A was zoonotic but lineage B was thought to be an ancestral genotype which is not adapted to humans [18].Similarly, phylogenetic approaches utilized for Plasmodium parasites have provided insights regarding the significant variation in clinical disease across host species [19].These examples provide support for the importance of investigating the phylogenetic relationships of parasites.Our study is the first depositing cox1 sequences and haplotypes from the United States.
The present study provides a basic understanding of T. canis haplotypes in the United States, but much could be revealed through more extensive sampling.Genetic studies of many veterinary parasites are conducted as specimens are obtained opportunistically; collecting adult worms is logistically challenging for a variety of reasons.Also, as Fava [10] hypothesized, movement of animals and therefore their parasites could be influencing the phylogeographic results measured by DNA sequencing data.While the approximate location of the animal at the time of specimen collection was recorded and utilized for the present analysis, no information regarding previous travels of the animals was collected.Similarly, our state-level data is not amenable to a precise analysis.Still, this study demonstrated that even opportunistic sampling reveals interesting traits about the haplotypes found in the United States and globally.
The cox1 gene has been described as a more ideal target for phylogeographic analysis due to a relatively high mutation rate, intraspecific variability, and maternal inheritance [20].In this  PLOS NEGLECTED TROPICAL DISEASES study, we expected to find new cox1 haplotypes since there is a paucity of data available for North America.However, this was not the case: our results suggest that haplotypes reported abroad were also present in the United States.Moreover, analysis of median joining networks and maximum likelihood analysis of consensus haplotypes both sorted parasites into five main clades.Interestingly, 51/55 haplotypes fell in the same predicted clades in both analyses.Thus, our results suggest that populations of T. canis with specific characteristics may be distinguishable on a global scale.Whether or not these populations represent cryptic species is a matter of speculation at this time, we refrain from naming them with any significant nomenclature.While our finding of specific clades of T. canis is enticing, further clinical or life cycle traits can be correlated with our sequence-based clades.Indeed, such labels should only be pursued if they are useful for scientists and clinicians [21].While our finding of specific clades of T. canis is enticing, one limitation of the study was the lower bootstrap support in the maximum likelihood trees which indicates a decreased amount of variation in this gene from the population under study.A different mitochondrial gene, such as nad4, or analysis of multiple genes may provide a higher level of detail to allow for the elucidation of phylogeographic relationships that may exist [22].Indeed, a concatenation of data from multiple genes can be used to clarify the picture of nematode phylogeny [23] In the present study, we investigated populations of T. canis from the United States.Sequencing of cox1 sequences enabled us to deposit new GenBank sequences from specimens in the United States and make comparison with those available worldwide.Using analysis, we found that cox1 sequences were moderately supportive of clustering, whereby 5 clades were present throughout the world.However, this clustering could not be explained solely by geography.Additional studies are needed to determine if the various haplotypes have unique life history or clinical traits worthy of attention by clinicians.In summary, this study contributes a broader understanding of T. canis haplotypes and provides a basis for future genetic studies of the parasite that identify zoonotic haplotypes found in human infection.

Fig 1 .
Fig 1. Maximum likelihood using T. canis samples collected in this study from the United States.Branches with greater than 50% bootstrap support are indicated with the corresponding bootstrap value.https://doi.org/10.1371/journal.pntd.0011665.g001

Fig 2 .
Fig 2. Maximum likelihood using both sequences from this study and global sequences available on Genbank.Branches with greater than 50% bootstrap support are indicated with the corresponding bootstrap value.https://doi.org/10.1371/journal.pntd.0011665.g002

Fig 3 .
Fig 3. Haplotype consensus maximum likelihood tree from all available T. canis cox1 sequences in GenBank; haplotypes are given in parentheses.Haplotypic clusters are colored to match the median joining network in Fig 4. Branches with greater than 50% bootstrap support are indicated with the corresponding bootstrap value.https://doi.org/10.1371/journal.pntd.0011665.g003

Fig 4 .
Fig 4. Median joining network of T. canis haplotypes found worldwide.Haplotypic clusters are colored to match the maximum likelihood tree in Fig 3. Colored circles represent individual haplotypes.Hash marks indicate the number of nucleotide differences between different haplotypes.https://doi.org/10.1371/journal.pntd.0011665.g004